For the investigation of biological specimens, it has been usual for some time to prepare the specimen using optical markers, in particular fluorescent dyes. Often, for example in the field of genetic research, several different fluorescent dyes are introduced into the specimen and become attached specifically to certain specimen constituents. From the fluorescence properties of the prepared specimen conclusions can be drawn, for example, as to the nature and composition of the specimen or the concentration of certain substances within the specimen.
In scanning microscopy in particular, methods that exploit location-dependent nonlinearities of the specimen are used. This field includes, for example, coherent anti-Stokes Raman scattering (CARS), which is known inter alia from PCT Application WO 00/04352 A1. It must be noted, however, that illuminating light having at least two different illuminating light wavelengths, at high light power levels, is required for this method.
Another method that makes use of nonlinear effects is so-called STED (stimulated emission depletion) microscopy, known for example from PCT/DE/95/00124. Here the lateral edge regions of the focus volume of the excitation light beam are illuminated with a light beam of another wavelength, called the stimulation light beam, that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, resulting overall in improved resolution.
In multi-photon scanning microscopy, the fluorescent photons attributable to a two-photon or multi-photon excitation process are detected. The probability of a two-photon transition depends on the square of the excitation light power level, and therefore occurs with high probability at the focus of the scanning illuminating light beam, since the power density is highest there. To achieve sufficiently high light power levels, it is useful to pulse the illuminating light and thereby achieve high peak pulsed light power levels. This technique is known, and is disclosed e.g. in U.S. Pat. No. 5,034,613 “Two-photon laser microscopy” and in German Unexamined Application DE 44 14 940. A further advantage of multi-photon excitation especially in confocal scanning microscopy lies in the improved bleaching behavior, since the specimen bleaches out only in the region of sufficient power density, i.e. at the focus of an illuminating light beam. Outside that region, in contrast to one-photon excitation, almost no bleaching takes place.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To allow acquisition of image data in layers, the specimen stage or the objective is displaced after a layer has been scanned, thus bringing the next layer to be scanned into the focal plane of the objective.
Spectral influencing of light pulses by amplitude modulation or phase modulation is known from the literature, e.g. from Rev. of Scientific Instruments 71 (5) pp. 1929–1960. Spectral modification of the laser pulses is usually used to shorten the pulses, to shape them optimally, or to control optically induced processes.
The aforesaid methods are disadvantageous in that high light power levels are necessary, resulting on the one hand in great demands on the light source and on the other hand in undesirable damage to the specimen, for example due to bleaching.